Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family
Abstract
:1. Introduction
2. Results and Discussion
2.1. Thermal Shift Assay of ARC/PIM Complexes
2.2. Co-Crystal Structures of ARC-1411, ARC-1415, and ARC-3126 with PIM-1
2.3. Design and Biochemical Characterization of New Inhibitors
2.4. Kinome-Wide Selectivity Profiling
2.5. Surface Sandwich Assay for the Measurement of PIM-2 Concentration
2.6. Cell Plasma Membrane-Penetrative Properties of Dye-Labeled ARCs
3. Materials and Methods
3.1. Materials
3.2. Synthesis of Compounds
3.3. Thermal Shift Assay for ARC/PIM Complexes
3.4. Co-Crystallization, Diffraction Data Collection, and Structure Determination
3.5. Biochemical Binding/Displacement Assays with Measurement of FA or TGLI
3.6. Kinase Selectivity Profiling
3.7. Surface Sandwich Assay for PIM-2 Measurement
3.8. Cell Culture, Transfection, and Imaging
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Compound | Structure | ∆Tm, °C | ||
---|---|---|---|---|
PIM-1 | PIM-2 | PIM-3 | ||
ARC-684 | PYB-Ahx-d-Arg-Ahx-(d-Arg)6-d-Lys-NH2 | 7.4 ± 0.2 | 7.7 ± 0.5 | 6.3 ± 0.3 |
ARC-668 | AMTH-Ahx-d-Arg-Ahx-(d-Arg)6-d-Lys-NH2 | 6.7 ± 0.2 | 8.1 ± 0.6 | 6.9 ± 0.3 |
ARC-1141 | AMTH-Ahx-d-Ala-(d-Arg)6-d-Lys-Gly | 5.2 ± 0.4 | 5.9 ± 0.6 | 4.9 ± 0.4 |
ARC-1411 | PIPY-C(=O)-(CH2)7-C(=O)-(d-Arg)6-d-Lys-NH2 | 7.6 ± 0.3 | 8.4 ± 0.4 | 6.0 ± 0.2 |
ARC-3119 | BBTP-Hyp-Ahx-(d-Arg)9-d-Lys-NH2 | 13.7 ± 0.4 | 13.9 ± 0.6 | 11.9 ± 0.2 |
ARC-3125 | TIBI-CH2-C(=O)-Ahx-(d-Arg)6-d-Lys-NH2 | 12.7 ± 0.2 | 14.7 ± 1.0 | 12.9 ± 0.2 |
SGI-1776 | Not shown | 9.5 ± 0.4 | 8.1 ± 0.6 | 8.2 ± 0.3 |
≡ BPTP ≡ BBTP | |||||
---|---|---|---|---|---|
Compound # | Structure a | KD, nM (PIM-1) b | KD, nM (PIM-2) b | KD, nM (PIM-3) b | KD, nM (PKAcα) b |
ARC-3126 | BPTP-Ahx-(d-Arg)6-d-Lys-NH2 | 1.8 ± 0.7 | 2.9 ± 0.5 | 2.0 ± 0.5 | >18,460 |
1 (ARC-2067) | BPTP-Aoc-(d-Arg)2-Gly-d-Arg-d-Lys-NH2 | 11.4 ± 1 | 64 ± 8 | 5.2 ± 1 | 2250 ± 411 |
2 (ARC-2059) | BBTP-Aoc-(d-Arg)2-Gly-d-Arg-d-Lys-NH2 | 0.4 ± 0.1 | 3.8 ± 0.2 | 2.0 ± 0.4 | 853 ± 28 |
3 (ARC-2060) | BBTP-Amt-(d-Arg)2-Gly-d-Arg-d-Lys-NH2 | 0.7 ± 0.2 | 9.9 ± 0.8 | 7 ± 1 | 1502 ± 266 |
4 (ARC-2061) | BBTP-Tap-(d-Arg)2-Gly-d-Arg-d-Lys-NH2 | 1.8 ± 0.3 | 20 ± 4 | 6.6 ± 1.4 | >18,210 |
5 (ARC-2062) | BBTP-Hyp-Aoc-(d-Arg)2-Gly-d-Arg-d-Lys-NH2 | 37.8 ± 9 | 447.4 ± 52 | 30.4 ± 6.5 | >20,590 |
6 (ARC-2074) | BBTP-Aoc-(d-Arg)2-Gly-d-Arg-[d-Lys(Cy5)]-NH2 | 1.7 ± 0.4 | 5.2 ± 0.9 | 2.6 ± 0.3 | >303 |
7 (ARC-2076) | BBTP-Tap-(d-Arg)2-Gly-d-Arg-[d-Lys(Cy5)]-NH2 | 21.9 ± 4 | 28.2 ± 6.7 | 8.3 ± 1 | >1076 |
8 (ARC-2065) | BBTP-Tap-(d-Arg)2-Gly-d-Arg-[d-Lys(5-TAMRA)]-NH2 | 20.5 ± 4 | 157 ± 39 | 15.7 ± 3 | >1020 |
PIM peptide | (d-Arg)2-Gly-d-Arg-d-Lys-NH2 | >11,490 | >46,020 | > 16,420 | >89,000 |
AZD1208 | Not shown | 5.3 ± 1 | 11.8 ± 2 | 2.3 ± 0.4 | >83,000 |
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Nonga, O.E.; Lavogina, D.; Enkvist, E.; Kestav, K.; Chaikuad, A.; Dixon-Clarke, S.E.; Bullock, A.N.; Kopanchuk, S.; Ivan, T.; Ekambaram, R.; et al. Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family. Molecules 2021, 26, 4353. https://doi.org/10.3390/molecules26144353
Nonga OE, Lavogina D, Enkvist E, Kestav K, Chaikuad A, Dixon-Clarke SE, Bullock AN, Kopanchuk S, Ivan T, Ekambaram R, et al. Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family. Molecules. 2021; 26(14):4353. https://doi.org/10.3390/molecules26144353
Chicago/Turabian StyleNonga, Olivier E., Darja Lavogina, Erki Enkvist, Katrin Kestav, Apirat Chaikuad, Sarah E. Dixon-Clarke, Alex N. Bullock, Sergei Kopanchuk, Taavi Ivan, Ramesh Ekambaram, and et al. 2021. "Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family" Molecules 26, no. 14: 4353. https://doi.org/10.3390/molecules26144353
APA StyleNonga, O. E., Lavogina, D., Enkvist, E., Kestav, K., Chaikuad, A., Dixon-Clarke, S. E., Bullock, A. N., Kopanchuk, S., Ivan, T., Ekambaram, R., Viht, K., Knapp, S., & Uri, A. (2021). Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family. Molecules, 26(14), 4353. https://doi.org/10.3390/molecules26144353